Merkel cell carcinoma (MCC) is an aggressive skin cancer with an

Merkel cell carcinoma (MCC) is an aggressive skin cancer with an increasing incidence. concentrations of NVP-BEZ235 for 12, 24, 48 and 72 h, respectively, and cell viability was analyzed using the CCK-8 assay. As shown in Fig. 2A, treatment with NVP-BEZ235 reduced the viability of MKL-1 cells in a time- and concentration-dependent manner. The ability of NVP-BEZ235 to modulate the signaling pathway in MKL-1 cells was then assessed by western blotting. As expected, NVP-BEZ235 markedly decreased the levels of Akt and mTOR phosphorylation in a dose-dependent manner in the assessed Vemurafenib MKL-1 cells (Fig. 2B), confirming the effect of NVP-BEZ235 on MCC. Figure 2. Vemurafenib NVP-BEZ235 Rabbit Polyclonal to DHRS4 treatment inhibits cell proliferation and attenuates the activity of the PI3K/Akt/mTOR signaling pathway. (A) Findings of the cell counting kit-8 assay revealing the sensitivity of MKL-1 cells to NVP-BEZ235. The cells were cultured with NVP-BEZ235 … NVP-BEZ235 induces cell cycle arrest, Vemurafenib but not apoptosis To further gain insight into the mechanisms of growth inhibition exerted by NVP-BEZ235, the effect of this agent on the cell cycle and apoptosis in MKL-1 cells was analyzed by flow cytometry. Consistent with the anti-proliferative effects of NVP-BEZ235, a pronounced decrease of cells in the S phase and a concomitant increase in cells in the G0/G1 phase were observed in the treated groups compared with the control group (Fig. 3A), indicating cell cycle arrest in the G0/G1 phase. Notably, NVP-BEZ235 did not induce apoptosis in MKL-1 cells, which was further confirmed by the absence of caspase-3 cleavage and activation (Fig. 3B and C). Collectively, these data indicated that NVP-BEZ235 induced G0/G1 cell cycle arrest, but not apoptosis, in MKL-1 cells. Figure 3. NVP-BEZ235 induces cell cycle arrest, but not apoptosis, in MKL-1 cells. (A) The proportion cells in each cell cycle phase in MKL-1 cells. The cells were treated with 100 nM NVP-BEZ235 for 24 h and stained with bromodeoxyuridine and 7-aminoactinomycin … Cell cycle arrest induced by NVP-BEZ235 is mainly dependent on p21 and p27 upregulation To explore whether cell cycle regulatory proteins were involved in the cell cycle arrest of MKL-1 cells, the cells were treated with various concentrations of NVP-BEZ235 and cell lysates were subjected to western blot analysis. As revealed in Fig. 3D, downregulation of the cell cycle promoter cyclin D1 and upregulation of the negative cell cycle regulators p21 and p27 were detected subsequent to NVP-BEZ235 treatment in MKL-1 cells. The MKL-1 cells were therefore transfected with shRNA targeting either p21 or p27 (Fig. 4A). As shown in Fig. 4B, knockdown of p21 or p27 expression partially rescued NVP-BEZ235-induced cell cycle arrest to a similar degree, which indicates that NVP-BEZ235-induced suppression of proliferation mainly results from the upregulation of p21 and p27. Figure 4. NVP-BEZ235-induced cell cycle arrest depends on the upregulation of p21 and p27. (A) Western blotting revealing the successful knockdown of p21 and p27 expression by shRNA in transfected MKL-1 cells. (B) Flow cytometry revealed a significant reduction … Discussion In the present study, the efficacy of NVP-BEZ235 as a potential therapeutic inhibitor of the PI3K/Akt/mTOR pathway was demonstrated in the human MCC MKL-1 cells. The results of the present analysis demonstrated that NVP-BEZ235 was effective in inhibiting proliferation and inducing cell cycle arrest in MKL-1 cells. Additional investigation revealed that NVP-BEZ235 attenuated PI3K/Akt/mTOR signaling and upregulated the expression of p21 and p27. Overall, these results have significant implications for the future development of dual PI3K/mTOR inhibitors as potential agents to treat human MCC. Deregulation of the PI3K/Akt/mTOR pathway is a common feature of numerous human cancers and contributes to cancer cell survival, promotes resistance to chemotherapy and radiotherapy through the disruption of apoptosis, and initiates cap-dependent translation of mRNA, which is essential for cell cycle progression, differentiation and growth (16C18). By employing the human MCC samples, the present study also confirmed the activation of PI3K/Akt/mTOR signaling in MCC, which was consistent with previous results (8). Consequently, it is.